JOURNAL OF VIROLOGY, Sept. 1992, p. 5598-5602

Vol. 66, No. 9

0022-538X/92/095598-05$02.00/0 Copyright X 1992, American Society for Microbiology

Multimerization of ICPO, a Herpes Simplex Virus Immediate-Early Protein JIANXING CHEN, CHRISTOS PANAGIOTIDIS, AND SAUL SILVERSTEIN* Department of Microbiology, Columbia University, 701 West 168th Street, New York, New York 10032 Received 3 April 1992/Accepted 22 May 1992 ICPO, a herpes simplex virus immediate-early gene product, is a highly phosphorylated nuclear protein that is a potent activator of virus and host genes. Using biochemical and genetic assays employing plasmids encoding mutant forms of ICPO and a recombinant adenovirus that expresses ICPO, we provide evidence that the protein multimerizes. Some mutant forms of ICPO were transdominant and interfered with activation of a target reporter gene or with complementation of an ICPO-minus virus.

mentation properties consistent with it being a multimer (12). To confirm this finding, the elution profile of native ICPO synthesized in mammalian cells in the absence of other herpes simplex virus proteins was examined. In this study, nuclear extracts were prepared (7, 29) from HeLa cells infected with MLP-0 (an adenovirus recombinant that produces biologically active ICPO [41]) for 24 h and the soluble proteins were concentrated by (NH4)2SO4 precipitation. The precipitate was dissolved and chromatographed on a Sephacryl-S400 column along with appropriate size markers. Fractions containing ICPO were identified by a dot blot procedure with a polyclonal antiserum prepared against a bacterial fusion protein. The profile of ICPO elution from this column is shown in Fig. 1. The native protein elutes just after the void volume, slightly behind a 658-kDa marker. This position is consistent with the protein either being complexed with a host protein or existing as a multimer. Monomeric ICPO would be expected to elute just after the carbonic anhydrase marker in fractions 27 to 32. In these experiments, we found no evidence of immunoreactive material at any position other than that indicated in Fig. 1. When extracts were prepared from cells infected with HSV-1, ICPO eluted in the same position, just before ICP4, a 175-kDa protein known to exist as a dimer (25, 35) (data not shown). ICPO could be in a complex with a host protein, but this would require the cell protein to be extremely abundant, because the peak is sharp and the elution profile does not change with the abundance of ICPO. The Sephacryl-S400 elution profile of ICPO is consistent with the size and sedimentation properties of ICPO synthesized in insect cells by a recombinant baculovirus (12) and suggests that the protein is present as an oligomer. A plasmid encoding ICPO will transactivate a reporter cassette driven by a herpes simplex virus promoter when cotransformed into mammalian cells (8, 15, 16, 27, 28). We previously demonstrated that tsK, an IE-4 gene mutant, exhibited a dominant negative phenotype when coinfected with wild-type virus at relatively high ratios of mutant to wild-type virus (15). To determine whether any of the transactivation-deficient IE-0 mutants that we isolated displayed a dominant negative phenotype, Vero cells were cotransfected with equimolar amounts of a construct expressing wild-type ICPO (pXQ1) and a tk-lacZ reporter and various molar-gene equivalents of three mutants that were defective in transactivation of the tk promoter or a wild-type control. The plasmids used in these experiments, along with the location of each mutation, are diagrammed in Fig. 2, and

Herpes simplex virus type 1 (HSV-1) encodes five immediate-early gene products. Two of these, ICP4 and ICP27, are obligatorily required for replication in tissue culture. ICPO, one of the other three, is a powerful transcriptional activator. IE-0, the gene encoding ICPO, is readily deleted from the virus and therefore is not required for growth in tissue culture. However, IE-0 deletion mutants replicate poorly. It is clear that ICPO facilitates replication of the virus, and a recent study demonstrated that ICPO augments virus gene expression in situations in which a-TIF, the virion-associated transactivator, is not present (3). Molecular dissection of this 775-amino-acid protein (30) by examination of the properties of insertion, nonsense, and deletion mutants (3, 6, 9-11) suggests that it is composed of several functional domains. These regions of the protein function in transcriptional activation of virus and cell promoters, functional interaction with ICP4 (16, 27, 34), nuclear localization (9), and perhaps interaction with a cell factor (39). Viruses with mutations in the IE-0 coding sequences have been isolated and characterized (3, 5, 11). These mutants have demonstrated that ICPO augments expression of virus genes from all of the temporally regulated classes of virus promoters (4, 5). This effect can be demonstrated at the level of accumulation of virus RNAs and proteins. The mechanism of how ICPO mediates gene activation is unknown. A recent report suggested that it induced expression of AP-1 (19). Parenthetically, we note that infection of undifferentiated F9 cells (which do not express AP-1 [21]) does not result in induction of AP-1 activity. Alternatively, ICPO could activate a cellular transcription factor (1), function as a bridge molecule in the manner of ot-TIF (20, 26, 32, 38), or suppress a negative regulatory factor (18, 33). Whether ICPO functions as a monomer or an oligomer has not been determined. To address some of these issues, we examined the elution profile of ICPO synthesized in mammalian cells on a sizing column. We also utilized plasmids containing mutations in the IE-0 coding sequence to determine whether they interfered with the activation potential of a wild-type IE-0 gene and whether they complemented replication of transfected virus DNA isolated from either the wild type or an IE-0 null mutant of HSV-1. Our results suggest that ICPO can function as an oligomer and that the N-terminal 104 amino acids serve this function. ICPO isolated from a recombinant baculovirus has sedi*

Corresponding author. 5598

VOL. 66, 1992

NOTES

5599

10

v VOID

V_ p

v 158

v 43kd

ICPO

0.4-

A595 -D07.8

e

7.6 :

0.3-

0.2

0a

0.1

0.

pCM1/2

.A.

*-

pCM89 -*-- pCM1 1/93

U1.

1..

u -

I.

10

20

30

40

50

I

w

60

FractonNkmber FIG. 1. Profile of elution of ICPO from Sephacryl-S400. Nuclear extracts concentrated by precipitation with (NH4)2SO4 were pre-

pared from cells infected with MLP-0. After resuspension in 10 mM Tris-HCl (pH 8.0)-i mM EDTA-0.01% CHAPS {3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate-0.1 mM TLCK (Na-p-tosyl-L-lysine chloromethyl ketone)-0.2 mM phenylmethylsulfonyl fluoride-10 mM 13-mercaptoethanol-300 mM (NH4)2SO4, the mixture was applied to a Sephacryl-S400 column. The elution profile of total protein was monitored by measuring the A595 of an aliquot of each fraction, and ICPO was detected by a dot blot procedure with antibody specific for ICPO. The elution profile of the protein markers (thyroglobulin [658 kDa], carbonic anhydrase [158 kDa], and ovalbumin [43 kDa]) was determined in multiple runs of the same column under identical conditions.

the results are shown in Fig. 3. Removal of the acidic N-terminal domain (pCM1/2; deletion of amino acids 12 through 104 [A12-1041) resulted in a twofold decrease in transactivation of a tk reporter and poor cooperation with ICP4 in activating the tk promoter (6). Cotransfection of pCM1/2 with pXQ1 resulted in no change in the level of ,B-galactosidase activity, at ratios of mutant to wild type ranging from 1:1 to 4:1. In contrast, two other mutants (pCM89 [in210/211] and pCM11/93 [A628-769]), each of

0

1 2 4 3 5 Ratio of EffectorPlasmids to Wild-Type

FIG. 3. Interference with activity of ICPO by mutant DNAs. Vero cells were cotransfected with 2.5 ,ug of pJC13 (tk-lacZ) and equimolar amounts of pXQ1 (wild-type IE-0) along with one-, two-, and fourfold molar-gene equivalents of the indicated mutants. The molar equivalents of IE-0 were normalized for each transfection by the addition of pIGA65 (16), a plasmid containing only the upstream promoter, regulatory, and leader sequences of IE-0. The DNA masses of the precipitates were adjusted to identical amounts by the addition of plasmid pIC2OR. The cells were shocked with 15% glycerin for 2 min at 24 h posttransfection. Fold stimulation over the basal level of pJC13 is shown. The background level of expression from pJC13 stimulated by each mutant has been subtracted. The datum points are averaged from at least two independent experiments, each done in duplicate.

which was more defective than pCM1/2 in the transactivation assay (6), dramatically inhibited induction of the tk reporter.

This assay was extended to determine whether other transactivation-defective mutants were dominant to wildtype ICPO (Fig. 3). The mutant pCM125, in which the insertion at position 124 disrupts the spacing in the Cys-rich IE-0 PLASMIDS is only partially dominant. Both pCM89 and pCM7 region, pxol (in212), which are transactivation deficient but whose mutapca tions are downstream of the Cys-rich region, are dominant. 1h86 169 ~~~~~~~~~~~~~~~~~pCA12 Four other defective deletion mutants were examined. pt pCM2/7 (A106-212), which has both the Cys-rich region and the adjacent downstream region deleted, is only partially pC11125 1Pi pCAZ2 dominant. Thus, removal of the region thought to be the 124~~~~~~~~~~~~~~pH2 major transactivation domain (5, 6, 9, 10) did not result in as severe a dominant phenotype as some of the codon insertion 212 mutations. In contrast, each of the other mutants with I pCNl7 overlapping mutations (pCM84/131 [A263-448], pCM88/131 449 2621 CB43 [A393-697], and pCM11/93 [A628-769]) was dominant to pC141131 ICPO. wild-type 392 697 The preceding experiment demonstrates that some of the ~~~~pCA0/31 399 463 defective transactivators can inhibit wild-type ICPO, whereas others cannot. There are several reasonable 628 hypotheses that might explain this result. Squelching, in pCSB + which increased ratios of mutant to wild-type forms of a 7I protein inhibit transcription by sequestering a transcriptional pCAfl 1 activator, is one mechanism that would result in this phenopCMI11/93 type (18). Another possibility is that one of the active forms in used IE-0 this FIG. 2. Locations of mutations in the of ICPO may result from multimerization. To examine study. The locations of codon insertion mutations and the boundwhether either of these mechanisms was responsible for the aries of the deletion mutations in plasmids encoding mutant forms of dominant phenotype, complementation and interference asICPO are shown. For details of the properties of each of these says were performed, with mutant and wild-type IE-0 plasmutants, see references 5, 6, and 40. 210211

i

697

628

769 [69

mutants

5600

J. VIROL.

NOTES TABLE 1. Interference with the synthesis of infectious virus by IE-0 mutantse

Cotransfected plasmid

pIGA65 pXQl pCM1/2 pCM2/7 pCM11/93

Virus titer 1.3 2.4 2.6 1.8 5.0

x x x x x

105 106

10 104 105

Fold

increase6

1.00 18.46 2.00 0.14 3.85

a2 x 106 Vero cells in 50-mm-diameter petri dishes were transfected approximately 20 h postseeding with 2 ,ug of infectious wild-type HSV-1 DNA and an approximately 40-fold molar excess of plasmid DNA encoding wildtype or mutant ICPO. The molar amounts of IE-0 were adjusted to the same amount for each transfection by the addition of pIGA65, which contains only the upstream promoter, regulatory, and leader sequences of IE-0, and the total amount of DNA present in each precipitate was adjusted to the same total concentration by the addition of plasmid pIC20R. Cultures were harvested at 56 h posttransfection, and virus yields on Vero cells were determined by plaque assay. Data represent two transfection experiments. b Fold increase = titer of cells cotransfected with mutant or wild-type ICPO/titer of pIGA65-cotransfected cells.

mids and naked virus DNAs from ICPO-plus and ICPO-minus viruses. A previous report demonstrated that cotransfection of virus DNA with an ICPO-expressing plasmid resulted in increased virus yield (3). Virus yields from cell cultures cotransfected with wild-type virus DNA and an IE-0 plasmid were measured after 56 h (Table 1). Control plasmids included pIGA65 (17), which contains only upstream promoter, regulatory, and leader sequences from the IE-0 gene, and pXQ1, which encodes wild-type ICPO. Wild-type ICPO stimulated yield about 20-fold and pCM1/2 and pCM11/93 marginally increased yield, whereas the product of pCM2/7 decreased virus yield by 7-fold. Thus, wild-type ICPO augments the yield from naked virus DNA in the absence of ao-TIF, the virion-associated transactivator of immediateearly genes (2, 31). The results with pCM1/2 and pCM2/7 were in line with those from the transactivation studies (Fig. 3 and 4); the plasmids either barely affected yield or decreased it, respectively. Surprisingly, pCM11/93 did not interfere with virus growth. This interference assay may be biased toward preferentially reflecting the activity of wildtype or mutant forms of ICPO on immediate-early gene promoters. In this regard, we note that pCM11/93 showed more activity on an immediate-early gene promoter than on the early gene promoter used in this study (6). Complementation analyses provide another approach to address how ICPO functions. Plasmids containing mutations in the IE-0 gene were cotransfected with DNA from HSV-1 d11403 (37), an IE-0 deletion mutant, and the virus yield was determined. A plasmid encoding wild-type ICPO complemented d11403 so that the yield was similar to that seen when only wild-type virus DNA is transfected (Fig. 5). Plasmids with insertion or deletion mutations were also tested. The data in Fig. 6 show virus yields at 72 h after cotransfection. A plasmid expressing wild-type ICPO increased the yield of d11403 by 1,000-fold over the baseline seen with only vector DNA (pIC20R) (Fig. 6A). Twenty-five different mutants were examined in this experiment, and the results with nine of these are shown. Twenty-two of the mutants increased virus yield at least 50-fold in the period examined. Of four mutants with alterations in the region spanning amino acids 124 to 213 (6), only pCM89 complemented d11403 to a significant extent. pCM89 is unique among the mutants we have studied; it is severely reduced in its ability to transactivate immediate-early and early gene promoters but rela-

20-

15O

1105-

0

C

IE-OPlasmid FIG. 4. Interference with activity of wild-type ICPO. Vero cells

were cotransfected with 2.5 ,ug of pJC13 (tk-lacZ) and equimolar amounts of pXQ1 (wild-type IE-0) along with a fourfold molar excess of the indicated mutants. The cells were shocked with glycerin at 24 h posttransfection, and ,-galactosidase activity was measured at 48 h posttransfection. Fold stimulation over the basal level of pJC13 is shown. The datum points are averaged from at least two independent experiments, each done in duplicate. The mutants are displayed across the abscissa according to the location of the mutation.

tively active on a late gene promoter (6). When moved to an adenovirus vector, pCM89 alone of all the transactivationdeficient IE-0 sequences reactivated latent herpes simplex virus in an in vitro latency model system (40). No increase in virus yield was seen when deletion mutant plasmids pCM1/2, pCM2/7, pCM84/131, and pCM88/131 were each cotransfected with virus DNA from d11403 (Fig. 6B). Note the difference in effect on virus yield when pCM2/7 is cotransfected with virus DNA from the wild type or d11403. In the instance in which a wild-type ICPO counterpart is available for interaction, virus yield is decreased

0

1

2

3

Days Post-transfection

4

FIG. 5. Complementation of dl1403 DNA by IE-0 sequences. Vero cells were cotransfected with 1 tg of infectious wild-type HSV-1 (strain 17) or dl1403 DNA, with or without 1 ,ug of pXQ1. The cells were shocked with glycerin at 24 h posttransfection. Cultures were harvested on days 1, 2, 3, and 4 posttransfection, and yields on Vero cells were determined by plaque assays as described

previously (5).

VOL. 66, 1992

NOTES

Insertion Mutants

7XTxU

A

mutant pCM2/7, with the major transactivation domain deleted, is not as dominant as mutants with codon insertions in this region are (Fig. 4 and Table 1). Assays of inhibition of transactivation by wild-type ICPO (Fig. 3 and 4) and of

*i

lxi

interference and/or complementation of virus yield after transfection are very different. Therefore, it remains difficult to explain the dominant nature of the carboxy-terminaldeletion mutants, because only half of them are able to augment replication of d11403 after transfection of naked virus DNA. We believe that the complementation and interference data rule out squelching as a mechanism of ICPOmediated gene activation because defective dominant mutants, such as pCM2/7, do not further decrease the yield of d11403 following cotransfection. There is no evidence of a specific interaction between promoter sequences from HSV-1 and ICPO, either alone or with another protein as for a-TIF (20, 26, 32, 38). Therefore, the potency of ICPOmediated transcriptional activation may result from transient interactions with a host protein(s) that regulates transcription or with RNA polymerase II itself. Our experiments do not rule out the possibility that the mutant forms of ICPO are transdominant because they specifically complex, and inactivate, a cell factor that normally interacts with ICPO. In this model, we would not expect to see a decrease in the yield of d11403. The experiments described above suggest that one of the active forms of ICPO is multimeric. We are currently further characterizing the purified protein and analyzing the function

1x103 1x102

1X100

CLL O1x106 FXm05

B

hI

DeletionMutants

ixi05

lXi

02

lxilO ixid'

IE-0 Complementing Plasmid FIG.

6.

Complementation

of d11403

DNA

by

5601

mutant

of the domains that constitute it. DNAs.

Vero cells were cotransfected with 1 ,ug of d11403 DNA and 1 ,ug of mutant plasmid DNA. The cells were shocked with glycerin at 24 h posttransfection. Cultures were harvested at 3 days posttransfection, and viral titers on Vero cells were determined by plaque assay. Complementation of d11403 by insertion mutants (A) and by deletion mutants (B) is shown. The mutants are displayed across the abscissa according to the location of the mutation.

Studies in the authors' laboratory were supported by Public Health Service grant GM38125 to S.S. from the National Institutes of Health. We are indebted to C. S. H. Young for helpful discussions of dominance and for critically reading the manuscript. We also thank the reviewer who suggested an alternative mechanism for transdominance by the mutant forms of ICPO.

below the basal level. In the other instance, in which there is no other source of ICPO, there is no decrease below the basal level. Although the other mutants examined (pCM131, pCM6, and pCM11/93) varied in their capacity to complement, all of them activated both immediate-early and early gene promoters to higher levels than any of the other deletion mutants examined for complementation (6). Transdominance assays have been used to define domains of virus regulatory molecules that are responsible for interacting with DNA or oligomerization (23, 24). A number of HSV-1 mutant proteins also display a dominant phenotype (13, 14, 16, 22, 35, 36). A recent study demonstrated that other IE-0 mutants were also transdominant (39). The experiments presented in this article provide biochemical and genetic evidence that one active form of ICPO is a multimer. The failure of pCM1/2, an N-terminal-deletion mutant, to dominate wild-type ICP0 but not complement d11403 is consistent with a model which predicts that the oligomerization domain maps to the region deleted from this mutant. This result is supported by studies showing that mutants with alterations in the major transactivation domain (amino acids 106 to 213) are dominant (Fig. 4) and do not complement (Fig. 6) but interfere with synthesis of infectious virus from a wild-type template (Table 1). We posit that these mutants dominate because they retain the ability to multimerize but poison the active oligomer with their defective subunit. If this line of reasoning is correct, it explains why

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